The Voltage-dependent Anion Channel Is the Target for a New Class of Inhibitors of the Mitochondrial Permeability Transition Pore
2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês
10.1074/jbc.m304748200
ISSN1083-351X
AutoresAndrea M. Cesura, Emmanuel Pinard, Robert Schubenel, Valérie Goetschy, Arno Friedlein, Hanno Langen, Peter Polčic, Michael Forte, Paolo Bernardi, John A. Kemp,
Tópico(s)Metabolism and Genetic Disorders
ResumoThe relevance of the mitochondrial permeability transition pore (PTP) in Ca2+ homeostasis and cell death has gained wide attention. Yet, despite detailed functional characterization, the structure of this channel remains elusive. Here we report on a new class of inhibitors of the PTP and on the identification of their molecular target. The most potent among the compounds prepared, Ro 68-3400, inhibited PTP with a potency comparable to that of cyclosporin A. Since Ro 68-3400 has a reactive moiety capable of covalent modification of proteins, [3H]Ro 68-3400 was used as an affinity label for the identification of its protein target. In intact mitochondria isolated from rodent brain and liver and in SH-SY5Y human neuroblastoma cells, [3H]Ro 68-3400 predominantly labeled a protein of ∼32 kDa. This protein was identified as the isoform 1 of the voltage-dependent anion channel (VDAC). Both functional and affinity labeling experiments indicated that VDAC might correspond to the site for the PTP inhibitor ubiquinone0, whereas other known PTP modulators acted at distinct sites. While Ro 68-3400 represents a new useful tool for the study of the structure and function of VDAC and the PTP, the results obtained provide direct evidence that VDAC1 is a component of this mitochondrial pore. The relevance of the mitochondrial permeability transition pore (PTP) in Ca2+ homeostasis and cell death has gained wide attention. Yet, despite detailed functional characterization, the structure of this channel remains elusive. Here we report on a new class of inhibitors of the PTP and on the identification of their molecular target. The most potent among the compounds prepared, Ro 68-3400, inhibited PTP with a potency comparable to that of cyclosporin A. Since Ro 68-3400 has a reactive moiety capable of covalent modification of proteins, [3H]Ro 68-3400 was used as an affinity label for the identification of its protein target. In intact mitochondria isolated from rodent brain and liver and in SH-SY5Y human neuroblastoma cells, [3H]Ro 68-3400 predominantly labeled a protein of ∼32 kDa. This protein was identified as the isoform 1 of the voltage-dependent anion channel (VDAC). Both functional and affinity labeling experiments indicated that VDAC might correspond to the site for the PTP inhibitor ubiquinone0, whereas other known PTP modulators acted at distinct sites. While Ro 68-3400 represents a new useful tool for the study of the structure and function of VDAC and the PTP, the results obtained provide direct evidence that VDAC1 is a component of this mitochondrial pore. The mitochondrial PTP 1The abbreviations used are: PTPpermeability transition poreIMMinner mitochondrial membraneOMMouter mitochondrial membraneVDACvoltage-dependent anion channelCyp-Dcyclophilin-DANTadenine nucleotide translocaseΔψmmitochondrial membrane potentialCsAcyclosporin AUb0ubiquinone 0Ub5ubiquinone 5FCCPcarbonyl cyanide p-trifluoromethoxyphenylhydrazoneBKAbongkrekic acidTFPtrifluoroperazineTPPtetraphenylphosphoniumATRatractylosideDIDS4,4′-diisothiocyanatostilbene-2,2′-disulfonic acidMALDI-MSmatrix-assisted laser desorption ionization-mass spectrometrynanoESI-MS/MSnanoelectrospray ionization tandem mass spectometryMOPS4-morpholinepropanesulfonic acid.1The abbreviations used are: PTPpermeability transition poreIMMinner mitochondrial membraneOMMouter mitochondrial membraneVDACvoltage-dependent anion channelCyp-Dcyclophilin-DANTadenine nucleotide translocaseΔψmmitochondrial membrane potentialCsAcyclosporin AUb0ubiquinone 0Ub5ubiquinone 5FCCPcarbonyl cyanide p-trifluoromethoxyphenylhydrazoneBKAbongkrekic acidTFPtrifluoroperazineTPPtetraphenylphosphoniumATRatractylosideDIDS4,4′-diisothiocyanatostilbene-2,2′-disulfonic acidMALDI-MSmatrix-assisted laser desorption ionization-mass spectrometrynanoESI-MS/MSnanoelectrospray ionization tandem mass spectometryMOPS4-morpholinepropanesulfonic acid. has been increasingly recognized as a major player in the mitochondrial pathways leading to cell death (1.Zoratti M Szabò I. Biochim. Biophys. Acta. 1995; 1241: 139-176Crossref PubMed Scopus (2188) Google Scholar, 2.Bernardi P. Physiol. Rev. 1999; 79: 1127-79155Crossref PubMed Scopus (1335) Google Scholar, 3.Crompton M. Biochem. J. 1999; 341: 233-249Crossref PubMed Scopus (2101) Google Scholar, 4.Bernardi P. Petronilli V. Di Lisa F. Forte M. Trends Biochem. Sci. 2001; 26: 112-117Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 5.Zamzami N. Kroemer G. ).Nat. Rev. Mol. Cell. Biol. 2001; 2: 67-71Crossref PubMed Scopus (887) Google Scholar). The PTP is triggered by Ca2+ influx into mitochondria and is modulated by a variety of factors that include mediators of intracellular signaling (2.Bernardi P. Physiol. Rev. 1999; 79: 1127-79155Crossref PubMed Scopus (1335) Google Scholar, 6.Gunter T.E. Gunter K.K. Sheu S.-S. Gavin C.E. Am. J. Physiol. 1994; 267: C313-C339Crossref PubMed Google Scholar, 7.Scorrano L. Penzo D. Petronilli V. Pagano F. Bernardi P. J. Biol. Chem. 2001; 276: 12035-12040Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). While it has been proposed that the PTP may provide mitochondria with a fast Ca2+ release channel, thereby contributing to intracellular Ca2+ homeostasis and signaling (2.Bernardi P. Physiol. Rev. 1999; 79: 1127-79155Crossref PubMed Scopus (1335) Google Scholar, 8.Ichas F. Jouaville L.S. Mazat J.P. Cell. 1997; 89: 1145-1153Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar, 9.Duchen M.R. J. Physiol. 2000; 529: 57-68Crossref PubMed Scopus (917) Google Scholar), persistent opening of the PTP precipitates a bioenergetic crisis with collapse of Δψm, ATP depletion, and Ca2+ deregulation. The PTP may also be instrumental in the release of mitochondrial apoptogenic proteins, such as cytochrome c, in programmed cell death (10.Newmeyer D.D. Ferguson-Miller S. Cell. 2003; 112: 481-490Abstract Full Text Full Text PDF PubMed Scopus (1074) Google Scholar). Several pieces of evidence suggest that mitochondrial dysfunction, associated with deregulation of the PTP, may play an important role in injury following ischemia/reperfusion (3.Crompton M. Biochem. J. 1999; 341: 233-249Crossref PubMed Scopus (2101) Google Scholar, 9.Duchen M.R. J. Physiol. 2000; 529: 57-68Crossref PubMed Scopus (917) Google Scholar, 11.Di Lisa F. Menabo R. Canton M. Barile M. Bernardi P. J. Biol. Chem. 2001; 276: 2571-2575Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar) and neuronal damage following an excitotoxic insult (12.Nicholls D.G. Budd S.L. Physiol. Rev. 2000; 80: 315-360Crossref PubMed Scopus (1046) Google Scholar). The PTP inhibitor CsA has been found to delay/reduce glutamate-induced mitochondrial membrane depolarization and cell death (13.Schinder A.F. Olson E.C. Spitzer N.C. Montal M. J. Neurosci. 1996; 16: 6125-6133Crossref PubMed Google Scholar, 14.Vergun O. Keelan J. Khodorov B.I. Duchen M.R. J. Physiol. 1999; 519: 451-466Crossref PubMed Scopus (199) Google Scholar) and to be neuroprotective in animal models of ischemia and brain trauma (15.Friberg H. Ferrand-Drake M. Bengtsson F. Halestrap A.P. Wieloch T. J. Neurosci. 1998; 18: 5151-5159Crossref PubMed Google Scholar, 16.Yoshimoto T. Siesjo B.K. Brain. Res. 1999; 839: 283-291Crossref PubMed Scopus (138) Google Scholar, 17.Okonkwo D.O. Povlishock J.T. J. Cereb. Blood Flow Metab. 1999; 19: 443-451Crossref PubMed Scopus (239) Google Scholar). Since non-immunosuppressive CsA analogues, such as N-MeVal4-CsA, have also been shown to have neuroprotective properties (18.Khaspekov L. Friberg H. Halestrap A. Viktorov I. Wieloch T. Eur. J. Neurosci. 1999; 11: 3194-3198Crossref PubMed Scopus (104) Google Scholar), this indicates that CsA is likely to act specifically to antagonize PTP dysfunction in these in vivo models. permeability transition pore inner mitochondrial membrane outer mitochondrial membrane voltage-dependent anion channel cyclophilin-D adenine nucleotide translocase mitochondrial membrane potential cyclosporin A ubiquinone 0 ubiquinone 5 carbonyl cyanide p-trifluoromethoxyphenylhydrazone bongkrekic acid trifluoroperazine tetraphenylphosphonium atractyloside 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid matrix-assisted laser desorption ionization-mass spectrometry nanoelectrospray ionization tandem mass spectometry 4-morpholinepropanesulfonic acid. permeability transition pore inner mitochondrial membrane outer mitochondrial membrane voltage-dependent anion channel cyclophilin-D adenine nucleotide translocase mitochondrial membrane potential cyclosporin A ubiquinone 0 ubiquinone 5 carbonyl cyanide p-trifluoromethoxyphenylhydrazone bongkrekic acid trifluoroperazine tetraphenylphosphonium atractyloside 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid matrix-assisted laser desorption ionization-mass spectrometry nanoelectrospray ionization tandem mass spectometry 4-morpholinepropanesulfonic acid. Despite detailed functional characterization, much of the information on the molecular nature of the PTP relies on indirect evidence and its precise molecular nature still remains elusive. The PTP is assumed to be due to the formation of dynamic multiprotein complexes at OMM and IMM contact sites (3.Crompton M. Biochem. J. 1999; 341: 233-249Crossref PubMed Scopus (2101) Google Scholar, 5.Zamzami N. Kroemer G. ).Nat. Rev. Mol. Cell. Biol. 2001; 2: 67-71Crossref PubMed Scopus (887) Google Scholar). These complexes are thought to involve the ANT in the IMM, in association with VDAC in the OMM, with a regulatory protein, Cyp-D, located in the matrix. Cyp-D is the target for CsA, and the only PTP regulatory protein identified so far with reasonable certainty (19.Nicolli A. Basso E. Petronilli V. Wenger R.M. Bernardi P. J. Biol. Chem. 1996; 271: 2185-2192Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar). Several other proteins, including those of the Bcl-2 family, appear to participate in PTP regulation through poorly defined interactions (5.Zamzami N. Kroemer G. ).Nat. Rev. Mol. Cell. Biol. 2001; 2: 67-71Crossref PubMed Scopus (887) Google Scholar). CsA has become the standard pharmacological tool for the characterization of the PTP in isolated mitochondria, in living cells and in vivo. Other PTP inhibitors include ANT ligands such as ADP and BKA (1.Zoratti M Szabò I. Biochim. Biophys. Acta. 1995; 1241: 139-176Crossref PubMed Scopus (2188) Google Scholar, 6.Gunter T.E. Gunter K.K. Sheu S.-S. Gavin C.E. Am. J. Physiol. 1994; 267: C313-C339Crossref PubMed Google Scholar), ubiquinone analogues (20.Fontaine E. Ichas F. Bernardi P. J. Biol. Chem. 1998; 273: 25734-25740Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 21.Fontaine E. Eriksson O. Ichas F. Bernardi P. J. Biol. Chem. 1998; 273: 12662-12668Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, 22.Walter L. Nogueira V. Leverve X. Heitz M.P. Bernardi P. Fontaine E. J. Biol. Chem. 2000; 275: 29521-29527Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), and a wide range of compounds, e.g. TFP and spermine, sharing the general property of being amphipathic cations (6.Gunter T.E. Gunter K.K. Sheu S.-S. Gavin C.E. Am. J. Physiol. 1994; 267: C313-C339Crossref PubMed Google Scholar, 23.Broekemeier K.M. Pfeiffer D.R. Biochemistry. 1995; 34: 16440-16449Crossref PubMed Scopus (208) Google Scholar). However, the fact that the molecular target for most of the compounds reported to inhibit or induce PTP have not been directly identified, together with their poor specificity, has hampered progress in elucidating PTP structure and pathophysiological relevance. In this article, we report the identification of a new class of inhibitors of the PTP. We also took advantage of the fact that these compounds carry a reactive moiety to perform affinity-labeling experiments for identifying its binding protein on the PTP. These studies led to the identification of the isoform 1 of VDAC (VDAC1) as their molecular target, providing direct evidence that this protein is indeed a component of the PTP. Compounds and Chemicals—Ro 04-2843 (6-bromo-3-diethylamino-methyl-chroman-4-one), Ro 68-3406 (6-bromo-3-methylene-chroman-4-one), Ro 68-3400 (spiro[cyclopentane-1,5′-[5H]dibenzo[a,d]cyclohepten]-2-one,10′,11′-dihydro-3-methylene) (see Fig. 1 for structures) were prepared at Hoffmann-La Roche Ltd, Basel, Switzerland. [3H]Ro 683400 (65 Ci/mmol, 1 Ci = 37 Mbq) was kindly prepared by Dr. Thomas Hartung (Hoffmann-La Roche Ltd). [3H]Tetraphenylphosphonium ([3H]TPP, 24–29 Ci/mmol) was purchased from Amersham Biosciences (Switzerland). CsA, TFP, Ub0, and Ub5 were from Sigma; ATR and BKA from BioMol; Calcium-Green 5N, Rhodamine-123, and DIDS from Molecular Probes. Preparation of Rat Liver Mitochondria—Liver and brain mitochondria were prepared from male Albino RoRo rats or MoRo mice (RCC, Basel, Switzerland). For swelling experiments, liver mitochondria were isolated by differential centrifugation according to standard procedures (21.Fontaine E. Eriksson O. Ichas F. Bernardi P. J. Biol. Chem. 1998; 273: 12662-12668Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). Brain mitochondria were isolated from rat and mouse forebrain on a Percoll gradient as described in Ref. 24.Sims N.R. J. Neurochem. 1990; 55: 698-707Crossref PubMed Scopus (340) Google Scholar. For affinity labeling experiments also liver mitochondria were purified on a Percoll gradient. Mitochondrial Swelling—Ca2+-induced swelling in energized mitochondria was assayed at 25 °C in 96 well-plates by measuring changes in absorbance at 540 nm by means of a SPECTRAMax 250 spectrophometer controlled by the SOFTmax PRO™ software (Molecular Devices). The incubation medium contained 0.2 m sucrose, 10 mm Tris-MOPS, 1 mm Pi-Tris, 5 μm EGTA-Tris, pH 7.4. Succinate (5 mm, in the presence of 2 μm rotenone) or 5 mm glutamate and 2.5 mm malate, buffered to pH 7.4 with Tris, were used as respiratory substrates. After a ∼5 min preincubation with or without test compounds, swelling was induced by the addition of 20 μl of CaCl2 at final concentrations ranging from 40 to 80 μm. The final incubation volume was 0.2 ml and the concentration of mitochondria was ∼0.5 mg mitochondrial protein/ml. Absorbance readings were taken every 12 s and the plate was shaken for 3 s between readings to ensure O2 diffusion. Swelling experiments were also performed in fully de-energized liver mitochondria according to Ref. 25.Chernyak B.V. Bernardi P. Eur. J. Biochem. 1996; 238: 623-630Crossref PubMed Scopus (210) Google Scholar. EC50 values were determined from dose-response curves using at least 7 different inhibitor concentrations. Data, expressed as percentage changes in absorbance at 540 nm (ΔA540) versus baseline (no CaCl2) 30 min after the addition of CaCl2, were fitted to non-linear regression analysis using a four-parameter logistic equation using the SigmaPlot computer program. [3H]TPP Uptake and Oxygen Consumption—Isolated liver mitochondria (∼0.5 mg protein/ml) were incubated in a batch mode in the presence of 20 nm [3H]TPP for 15 min at 25 °C. Aliquots (100 μl) of the mixture were then distributed into 96-well plates containing 100 μl of the test compound and the incubation prolonged for 15 min at 25 °C. Samples were then filtered through 0.3% (v/v) polyethyleneimmine-treated GF/B glass fiber filters using a 96-channel cell harvester and the filters washed twice with 1 ml of buffer. 50 μl of MICROSCINT 40 (Packard) were then added to each well, before counting for radioactivity in a TopCount scintillation counter (Packard). Nonspecific uptake was determined in the presence of 1 mm unlabeled TPP or 1 μm FCCP. Oxygen consumption was determined polarographically using a Clark-type electrode Measurement of Ca2+Retention Capacity and of Δψm in Brain Mitochondria—Extramitochondrial Ca2+ was determined using a PerkinElmer LS-50B fluorimeter controlled by the FL WinLab computer program. The incubation medium contained 0.2 m sucrose, 1 mm Pi-Tris, 10 mm Tris-MOPS, 5 mm glutamate-Tris, 2.5 mm malate-Tris, pH 7.4, containing 0.01% (w/v) bovine serum albumin, and 1 μm of the Ca2+ indicator Calcium Green-5N (21.Fontaine E. Eriksson O. Ichas F. Bernardi P. J. Biol. Chem. 1998; 273: 12662-12668Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). The final volume was 2.5 ml, and the cuvette was thermostatted at 25 °C. Brain mitochondria were subjected to a train of 5 μm Ca2+ additions and fluorescence monitored at excitation/emission wavelengths of 505–535 nm. Calibration of Ca2+ signals was performed according to the manufacturer's instructions assuming a KD for the dye of 14 μm. For measurements of Δψm, 0.5 μm rhodamine-123 was added instead of Calcium Green-5N and fluorescence monitored at 503–525 nm excitation/emission wavelengths (20.Fontaine E. Ichas F. Bernardi P. J. Biol. Chem. 1998; 273: 25734-25740Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Affinity Labeling of Mitochondria Using [3H]Ro 68-3400 —Percoll-purified mitochondria (∼30 μg of protein per sample) were incubated for 15 min at 25 °C in the presence of 10 nm [3H]Ro 68-3400 in a final volume of 200 μl. After rinsing, mitochondria were solubilized in sample buffer containing β-mercaptoethanol (1 h at 37 °C) and subjected to SDS-PAGE on Tris-glycine Novex precast gels (12% monomer concentration, Invitrogen). For fluorography, gels were soaked in Amplify™ (Amersham Biosciences), dried, and exposed at –80 °C to x-ray BioMax MS film with BioMax MS intensifying screen (Kodak). Protein Purification and Identification—Mitochondria (∼5 mg of proteins) were labeled as described above in the presence of 20 nm [3H]Ro 68-3400 for 15 min at 25 °C, and solubilized with 3 ml of 3% Triton X-100 (Surfact-Amps X-100, Pierce) in 10 mm NaPO4, pH 6.8, containing 0.5 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1.8 μg/ml aprotinin, and 1 μg/ml pepstatin A (Roche Applied Science). Samples were then injected into a ceramic hydroxyapatite CHT-II 1 × 5 cm column (Bio-Rad) equilibrated in 10 mm NaPO4, pH 6.8, containing 0.3% Triton X-100. The column was then eluted with NaPO4 gradient, containing 0.3% Triton X-100, at a flow rate of 0.5 ml/min. Fractions (1 min) were collected and, an aliquot (5 μl) counted for radioactivity, and then subjected to SDS-PAGE, followed by staining and fluorography. Proteins in the radioactive fractions were precipitated with trichloroacetic acid and, after carboxamidomethylation, submitted to SDS-PAGE. After staining with colloidal Coomassie Blue (Novex) and destaining, gel spots were excised and protein analyzed after in-gel digestion using modified trypsin (Promega) by MALDI-MS as previously described (26.Fountoulakis M. Langen H. Anal. Biochem. 1997; 250: 153-156Crossref PubMed Scopus (221) Google Scholar). Samples were analyzed in a time-of-flight PerSeptive Biosystems mass spectrometer equipped with a reflector. The peptide masses obtained were matched with the theoretical peptide masses of all proteins from all species in the SWISS-PROT and TrEMBL data base (us.expasy.org/sprot/). The identity of some of the tryptic fragments was also confirmed by nanoelectrospray ionization tandem MS (nanoESI-MS/MS) by means of an API 365 triple quadrupole mass spectrometer (Sciex) as previously described (27.Krapfenbauer K. Berger M. Friedlein A. Lubec G. Fountoulakis M. Eur. J. Biochem. 2001; 268: 3532-3537Crossref PubMed Scopus (41) Google Scholar). Yeast Methods—Saccharomyces cerevisiae yeast strains lacking the Por1 gene (Δpor1) and containing plasmids mediating the expression of yeast (YVDAC1) and human VDAC1 (HVDAC1) were produced and mitochondria prepared as described (28.Blachly-Dyson E. Zambronicz E. Yu W-H. Adams V. McCabe E. Adelman J. Colombini M. Forte M. J. Biol. Chem. 1993; 268: 1835-1841Abstract Full Text PDF PubMed Google Scholar, 29.Blachly-Dyson E. Song J. Wolfgang W.J. Colombini M. Forte M. Mol. Cell. Biol. 1997; 17: 5727-5738Crossref PubMed Scopus (139) Google Scholar, 30.Gross A. Pilcher K. Blachly-Dyson E. Basso E. Jockel J. Bassik M.C. Korsmeyer S.J. Forte M. Mol. Cell. Biol. 2000; 20: 3125-3136Crossref PubMed Scopus (145) Google Scholar). Affinity labeling experiments were performed in the presence of 10 nm [3H]Ro 68-3400 as described above. A mouse monoclonal antibody directed against HVDAC1 [anti-porin 31HL (Ab-4), Calbiochem], and a rabbit polyclonal antibody against YVDAC1 (29.Blachly-Dyson E. Song J. Wolfgang W.J. Colombini M. Forte M. Mol. Cell. Biol. 1997; 17: 5727-5738Crossref PubMed Scopus (139) Google Scholar) were used for VDAC detection by immunoblot in yeast mitochondrial preparations. Screening and Identification of a New Class of PTP Inhibitors—In an effort to identify new inhibitors of the PTP, a compound library was screened using Ca2+-induced swelling of rat liver mitochondria energized with succinate as a functional assay. Compounds found to inhibit swelling were then counter-screened using uptake of the potentiometic probe [3H]TPP (31.Hoek J.B. Nicholls D.G. Williamson J.R. J. Biol. Chem. 1980; 255: 1458-1564Abstract Full Text PDF PubMed Google Scholar) to discard "false positives" (e.g. protonophores), which could have lowered the mitochondrial membrane potential and, thus, Ca2+-influx into mitochondria. Compounds that did not interfere with mitochondrial respiration at the concentrations inhibiting the PTP were then selected for further characterization. Among these substances, our attention focused on a class of compounds inhibiting the PTP in the low micromolar range and having common pharmacophoric elements. These compounds were β-aminoketone derivatives, typically exemplified here by Ro 04-2843 (Fig. 1 and Table I). However, as also reported for a class molecules of close chemical structure (32.Ward E.F. Garling D.L. Buckler R.T. Lawler D.M. Cummings D.P. J. Med. Chem. 1981; 24: 1073-1077Crossref PubMed Scopus (48) Google Scholar), compounds with this structure are not stable at neutral pH, undergoing decomposition with t½ of ∼30 min at pH 7.4. The corresponding enone decomposition product of Ro 04-2843, i.e. Ro 68-3406 (Fig. 1), was, therefore, prepared and found to still inhibit PTP (Table I and Fig 2B). Preparation of a number of derivatives led then to the identification of a number of PTP inhibitors, including Ro 68-3400 (Fig. 1), which was identified as being the most potent.Table IEffect of various inhibitors on Ca2+-induced PTP in rat liver mitochondriaEC50nμmRo 04-28431.83 ± 0.39(3)Ro 68-34061.21 ± 0.43(6)Ro 68-34000.19 ± 0.03(3)CsA0.30 ± 0.03(4)Ubo23.2 ± 2.3(4)ADPaThe experiments with ADP were performed in the presence of 1 μg/ml oligomycin.4.84 ± 0.73(3)BKA11.5 ± 4.21(4)TFP9.37 ± 3.32(3)a The experiments with ADP were performed in the presence of 1 μg/ml oligomycin. Open table in a new tab Properties of Ro 68-3400 as PTP Inhibitor in Liver Mitochondria—The inhibition of Ca2+-induced swelling in liver mitochondria energized with NADH-linked substrates (glutamate/malate) by Ro 68-3400 is shown in Fig. 2A. This compound inhibited PTP induced by addition of 40 μm Ca2+ with an EC50 of 98 ± 10 nm (Fig. 2B). Under similar conditions, CsA and Ro 68-3406 displayed EC50 values of 160 ± 9 and 930 ± 30 nm, respectively (Fig. 2B). Table I shows the EC50 obtained in succinate-energized mitochondria for Ro 68-3400 and related compounds in comparison to known PTP inhibitors. Ro 68-3400 was at least as effective as CsA at inhibiting PTP in liver mitochondria, and more potent than the other PTP inhibitors tested. While it has to be pointed out that the EC50 are relative values and depend on Ca2+ load as well as on respiratory substrates (see Ref. 20.Fontaine E. Ichas F. Bernardi P. J. Biol. Chem. 1998; 273: 25734-25740Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar), the relative potencies of the various inhibitors were maintained at varying Ca2+ loads (data not shown). 2A. M. Cesura, E. Pinard, R. Schubenel, N. Hauser, V. Goetschy, E. Milanesi, P. Bernardi, and J. A. Kemp, manuscript in preparation. Ro 68-3400 and Ro 68-3406 were also effective at inhibiting PTP in de-energized mitochondria, a condition where interaction with sites indirectly modulating PTP can be excluded (25.Chernyak B.V. Bernardi P. Eur. J. Biochem. 1996; 238: 623-630Crossref PubMed Scopus (210) Google Scholar, 33.Linder M.D. Morkunaite-Haimi S. Kinnunen P.K. Bernardi P. Eriksson O. J. Biol. Chem. 2002; 277: 937-942Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), with EC50 values of 0.37 and 2.8 μm, respectively (n = 2, 200 μm Ca2+). For comparison, under these conditions, the EC50 values of CsA and Ub0 were found to be 0.22 and 4.9 μm, respectively. At concentrations completely blocking PTP, Ro 68-3400 and Ro 68-3406 did not inhibit mitochondrial respiration (basal, ADP-induced and uncoupled), Ca2+ uptake by mitochondria or Cyp-D peptidyl prolyl cis-trans isomerase enzymatic activity (data not shown). Effect of Ro 68-3400 on PTP in Brain Mitochondria—Ro 68-3400 inhibited swelling of brain mitochondria with a potency in the range of that observed for liver mitochondria (Fig. 3A). The effect of PTP inhibitors in brain mitochondria was also investigated by subjecting mitochondria to a series of 5 μm Ca2+ pulses and by monitoring extramitochondrial Ca2+ or Δψm using fluorescent probes (21.Fontaine E. Eriksson O. Ichas F. Bernardi P. J. Biol. Chem. 1998; 273: 12662-12668Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). The effects of CsA and Ro 68-3400 in these experiments are shown in Fig. 3, B and C. Both Ro 68-3400 and CsA increased the ability of mitochondria to buffer Ca2+ until a threshold was reached at which no further Ca2+ could be taken up (Fig. 3B). At 1 μm (i.e. the maximal effective concentration observed for each inhibitor), both compounds approximately doubled the amount of Ca2+ taken up by brain mitochondria. Thus, control mitochondria were able to accumulate 530 ± 70 nmol of Ca2+/mg of protein, whereas, in the presence of 1 μm CsA and Ro 68-3400, the Ca2+-buffering capacity increased up to 1130 ± 80 and 1200 ± 120 nmol of Ca2+/mg protein, respectively (mean ± S.E., n = 3). The combination of Ro 68-3400 and CsA had an additive effect, mitochondria being able to accumulate up to 2050 ± 450 nmol of Ca2+/mg of protein. In agreement with the finding that Ro 68-3400 does not inhibit Cyp-D enzymatic activity, this indicates that Ro 68-3400 acts at a site in the PTP other than Cyp-D. Virtually identical results were obtained from experiments where Δψm was monitored after a series of Ca2+ additions (Fig. 3C). Each Ca2+ addition caused reversible decreases in Δψm, until triggering of PTP completely collapsed Δψm and no further fluorescence increase could be observed after addition of the protonophore FCCP. Similar experiments using other known PTP inhibitors also showed that the effect of Ro 68-3400 (and of Ro 68-3406) was additive with that of BKA, ADP, TFP, and tamoxifen, suggesting that Ro 68-3400 acts at a site that is different from those acted upon by these compounds (data not shown).2 The only exception was Ub0, a previously characterized PTP blocker (20.Fontaine E. Ichas F. Bernardi P. J. Biol. Chem. 1998; 273: 25734-25740Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar) for which no such additive effect was seen. Ro 68-3400 and Ubiquinone Derivatives—The lack of additive effect with Ub0 suggested that the binding site of Ro 68-3400 might be related to the ubiquinone site reported to modulate the PTP (21.Fontaine E. Eriksson O. Ichas F. Bernardi P. J. Biol. Chem. 1998; 273: 12662-12668Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, 22.Walter L. Nogueira V. Leverve X. Heitz M.P. Bernardi P. Fontaine E. J. Biol. Chem. 2000; 275: 29521-29527Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). To address this, we investigated whether Ub5, an ubiquinone derivative able to relieve the inhibitory effect of Ub0 (21.Fontaine E. Eriksson O. Ichas F. Bernardi P. J. Biol. Chem. 1998; 273: 12662-12668Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar), could similarly antagonize PTP inhibition by Ro 68-3400. As shown in Fig. 4A for rat liver mitochondria, Ub5 (50 μm) was able antagonize the inhibition by Ro 68-3400, shifting its EC50 from 290 nm to 2.4 μm (n = 2). Likewise, in rat brain mitochondria, inhibition of Ca2+-induced depolarization by 300 nm Ro 68-3400 was relieved by 50 μm Ub5 (Fig. 4B). Affinity Labeling of Mitochondria with [3H]Ro 68-3400: Identification of VDAC1 as a Component of the Permeability Transition Pore—The high potency displayed by Ro 68-3400 at inhibiting the PTP and the fact it contains a reactive moiety, prompted us to tritiate the compound and use it as affinity labeling probe. Fig. 5A shows the results obtained after labeling with 10 nm [3H]Ro 68-3400 of intact mitochondria isolated from rat brain and liver. A restricted number of proteins appeared to be labeled with a predominant band of ∼32 kDa present in both preparations. A protein of identical size was also primarily labeled in mitochondria from mouse brain and liver, and from SH-SY5Y human neuroblastoma cells (not shown). Increasing concentration of unlabeled Ro 68-3400 inhibited radioactivity incorporation into this band. Notably, its labeling did not reflect a higher relative abundance, since Coomassie Blue and/or silver staining did not reveal major protein bands that correspond to the labeled protein (see inset to Fig. 5B). This ∼32 kDa protein labeled by [3H]Ro 68-3400 in rat brain and liver mitochondria could be purified by a single FPLC chromatographic step using a hydroxyapatite column. As shown in Fig. 5B, most of the radioactivity eluted in the column front. SDS-PAGE analysis of these fractions, followed by silver staining and fluorography, showed the pr
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